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daughterless coordinates somatic cell proliferation, differentiation and germline cyst survival during follicle formation in Drosophila

John E. Smith, III, Craig A. Cummings{dagger} and Claire Cronmiller*

Department of Biology, University of Virginia, P.O. Box 400328, Charlottesville, VA 22904-4328, USA
{dagger} Present address: Department of Microbiology and Immunology, Stanford University, Stanford, CA 94305, USA



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  		Fig. 1. The da loss-of-function phenotype. (A) Diagram of the anterior end of an ovariole, including terminal filament (tf), subdivided germarium (with regions indicated), and early follicles of the vitellarium. Somatic cells (gray), polar cells (purple) and stalks are indicated. Wild-type (B,F), dalyh/da2 (C), dalyh/dalyh (D,E,G,H) ovaries were costained for Hts (Hu li tai shao; green) and Vasa (magenta). At eclosion most da null ovarioles (C,D) have no distinct interfollicular stalks; in 10% of ovarioles (E), the posteriormost follicle is separated by a stalk, but always at the expense of the follicular epithelium of the second germline cyst produced. (G) Older da mutant ovaries, despite extensive defects within the vitellarium, have typical germarium morphology. Interfollicular stalks do not form, even when sufficient somatic cells accumulate (arrow) adjacent to follicles with multiple cysts. (H) In more extreme examples, regions 2b and 3 are lost within the compound follicles that constitute the remainder of the ovariole. (I) A typical DAPI-stained dalyh ovariole. Images B-E are at the same scale, as are images F-H.

 


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  		Fig. 2. The effect of da on polar and stalk cell markers. (A) Enhancer trap B1-93F marks interfollicular stalks, presumptive stalk cells and terminal filament in wild type. In da7/das22 (B) and dalyh (C) ovaries the enhancer trap stains clusters of cells that are sometimes arranged into rope-like patterns. Expression of B1-93F is weaker in da mutants (C, arrowheads) relative to the overexposed terminal filament (C, asterisk). This enhancer trap is also expressed in degrading somatic cells often present at the posterior of da mutant ovarioles (B, asterisk). (D) In wild type, enhancer trap A101 stains polar cells in each follicle; initial expression may include up to 4 cells at either pole (3 shown in inset), but by stage 4 staining is limited to 2 cells at each pole. (E) In da7/das22 mutant ovaries, clusters of 3 or 4 somatic cells are found throughout ovarioles, apparently corresponding to ends of each cyst in a compound follicle. (F) In more severely disrupted da mutant ovarioles, a large number of A101-positive cells are found throughout a region that is anterior to extensive degradation. (G) In wild type, FasIII protein is found initially in all somatic cells in regions 2b and 3 of the germarium; levels subsequently diminish in all but the polar cells. (H-I) In da mutants, clusters of 3 or 4 FasIII-positive cells are found along compound follicles, corresponding to cyst poles.

 


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  		Fig. 3. Chromosomal da+ duplications produce excess somatic cells. Ovaries from 3X-da+ flies (B, C) have longer interfollicular stalks than wild-type ovaries (A). (D) Enlargement of the anterior end of ovariole shown in C. (E) Ovaries with the B231 duplication (which includes da+) also produce more follicles than wild type, as indicated by the number of previtellogenic follicles per ovariole and (C) increased ovariole length. Staining: Hts (green), Vasa (magenta).

 


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  		Fig. 4. Elevated da+ levels were achieved with several genotypes and environmental conditions: (A,B) homozygous dalyh with phsp70-da+, 6 days at 32°C; (D,E) phsp70-da+, 37°C pulses; (F-L) homozygous dalyh with DpB231. (A,B) High levels of da+ drive formation of ectopic stalks at the expense of epithelium; partially exposed germline cysts (A',B') have stalk-like somatic cells (demarcated with black dots in A, B) that stretch along the cyst. (D,E) Pulses of Da result in aberrant somatic behavior; new cysts are elongated and initially fail to separate from the germarium (asterisks, compare to wild type, C). (F-L) Elevated da levels result in germline cyst degradation. Cyst remnants with decreased Vasa are seen in regions 2b and 3 of the germarium (arrows, F,G), in newly formed follicles (arrow, H) and in long stalks (arrow, I). Degrading compound follicles, with extensive Vasa staining (I,L''') are identifiable by DAPI (inset I,L). (H-J) In germaria depleted of mature cysts, still-dividing germline cysts, normally confined to region 2a, spread posteriorly and become surrounded by somatic cells. A late interphase 4-cell cyst (arrowhead, J) in region 3 is identifiable by its fusome structure (de Cuevas and Spradling, 1998Go) and DAPI (data not shown). (K) Spreading germline cells occasionally fall out of the germline stem cell niche (gray arrowhead points to empty niche). (L-L''') DAPI staining (L) and (L'-L''') different focal planes through a germlineless germarium. Germarium (bracket) without any germline (L''') is identifiable by the terminal filament (asterisk in L). Staining: DAPI (grayscale), Hts (green), Vasa (magenta). Yellow scale bar: 20 µm; all images same magnification as G except I, J and K.

 


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  		Fig. 5. Visualization of the cyst progression checkpoint. Acridine Orange staining (A,A',B,C) and TUNEL (E,E',F) identify apoptotic germline cells in region 2 of the germarium. Vital Acridine Orange staining of wild-type germaria in M3 medium is visible as green (A) or red (A') fluorescence; signal is localized to region 2 of the germarium. (B-C) In phosphate buffer, where Acridine Orange signal is amplified, dalyh mutants (B) show dramatically reduced fluorescence relative to wild type (C). (B,C are shown at the same magnification; lines indicate the positions of the germaria for comparison.) (D) Apoptosis as measured by vital Acridine Orange staining of germaria from 2- to 4-day old adult wild-type and da mutant females. Blue numbers indicate total number of germaria scored; bars show the average percentage per female ±s.e.m. (n=11 for da7/das22, n=10 for all others). (E,E') TUNEL (green) labels cells at the region 2a/2b boundary; staining corresponds with decreased DAPI intensity (E''). (F,F') Reduced Vasa (magenta) identifies apoptotic cells as germline and corresponds with smaller punctate DAPI staining.

 


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  		Fig. 6. Genetic interactions with mutants in the JAK/STAT and EGFR pathways. DAPI-stained ovarioles from flies doubly heterozygous for da2 and: (A) hop2, (B,C) Stat92E1681, (D) grkHK36, (E) sose4G, (F) phlc110, and (G) rlSem. da2 also acts as a dominant enhancer of topIP02/top1 (H) and brnfs.107/brn1.6P6 (I,J). The ovary interaction phenotypes are indistinguishable from those of da alone, except for the round ovariole morphology with brn.

 


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  		Fig. 7. Genetic interactions with ectopic hh. (A) Ectopic expression of hh produces excess somatic cells and long stalks. (B,C) Ectopic hh phenotype is partially suppressed in a da2 heterozygous genotype; follicles are often compressed into an elongated shape (C). (D) The da mutant genotype (dalyh/da2) is completely epistatic to ectopic hh. (E-H) Elevated da+ (DpB231; dalyh) enhances the ectopic hh phenotype: (E) interfollicular cell number is increased (inset: loss of region 3 cyst characteristic of excess da+). (F) Frequent branched stalks usually terminate with otherwise detached follicles to form ‘lollipop-like’ structures. (G,H) Enlarged view of ‘lollipops’. Staining: Hts (green), Vasa (magenta), DAPI (grayscale).

 


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  		Fig. 8. An expanded model for follicle formation. Step 1: signaling by hh from the terminal filament (red) creates a somatic stem cell niche at the region 2a/2b boundary, conferring stem cell characteristics on 2 somatic cells (green). Step 2: somatic stem cells produce undifferentiated mesenchymal cells (green) that surround the germline cyst and compress it into a lens shape in region 2b Step 3: EGFR signaling from the germline (yellow) provides a continuous proliferation signal to somatic cells. Step 4: Dl signaling (blue) induces somatic cells between adjacent cysts in region 2b to initiate polar cell differentiation (purple stippling). Step 5: Somatic cells that physically contact only one germline cyst differentiate as cuboidal epithelial cells (beige). Step 6: differentiating polar cells provide a ‘booster’ proliferative signal, while recruiting remaining somatic cells in region 2b to form stalks (straight purple arrows); these could be distinct signals. In region 3 lateral inhibition (curved purple arrows) eventually refines the polar cell number to two. Step 7: differentiating stalk cells (orange stippling) converge and extend (arrows) between polar cells of adjacent cysts to form an interfollicular stalk. Steps 2, 3, 6 and 7 require discrete da+ functions.

 

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© The Company of Biologists Ltd 2002